| AimChemotherapy is one of the major means to treat malignant cancers; it largely increases the possibility of successful treatment of some cancers. But the occurrence of drug resistance renders cancer cells resistant to anticancer agents leading to the failure of chemotherapy, which we call drug resistance. In mammalian cells, multidrug resistance (MDR) develops from an initial single drug resistance, eventually leading to a broad cross-resistance to many structurally and functionally unrelated compounds. At the moment, the mechanism of MDR is widely studied. The most important and best-known mechanism is that MDR results from the overexpression of P-glycoprotein (P-gp), a 170-kDa membrane glycoprotein coded by the mdr1 gene. Inhibition or modulation of the function and/or expression of P-gp is considered a realistic reverse approach to the P-gp mediated MDR. Cepharanthine is a natural bisbenzylisoquinoline alkaloid extracted from the roots of Stephania cepharantha Hayata, which has various strong biological activities in vitro and in vivo. Its reversing effect on multidrug resistance has been proved in studies abroad. However, the correlation between its effect on MDR and P-glycoprotein has not been reported. This study investigates the methods of cytology and molecular biology to determine the reversing effects of Cepharanthine Hydrochloride (CH) and its molecular mechanism against MDR.MethodsK562, a human chronic myelogenous leukemia cell line and its adriamycin-resistant variant K562/ADR cell line were used for the test. Cytotoxicity of ADR (adriamycin) and ADR combined with CH or v VER (Verapamil) in K562 and K562/ADR cells were determined by MTT (3-(4,-5-dimethylthiazol)-2,-5-diphenylte-2H-tetrazolium bromide) assay. Based on flow cytometric technology, the effect of CH or VER on the uptake and efflux of rhodamine123 (Rho123) and the accumulation of adriamycin in these cells were detected by measuring Rhl23 or adriamycin-associated mean fluorescence intensity (MFI). The effect of CH on P-glycoprotein expression in K562 and K562/ADR cells was also measured using a flow cytometer with PE-conjugated P-glycoprotein antibody. The aims of this study are to elucidate the relationship between drug resistance of K562/ADR to ADR and the function and expression of P-glycoprotein, as well as the mechanism of CH reversing drug resistance.Results1. Cytotoxity of ADR in K562 and K562/ADR cell linesThe IC50 of ADR in K562 and K562/ADR cell lines as determined by MTT assay were 0.45±0.01μmol·L-1 and 13.97±0.30μmol·L-1, respectively, with significant differences from each other at P<0.05 and the resistance times were 31.04.2. Cytotoxity of ADR combined with CH or VER in K562 and K562/ADR cell linesCytotoxity of CH or VER alone in K562 and K562/ADR cell lines was weak. The inhibitory rates (IR) of K562 and K562/ADR cells treated with 4μmol·L-1 CH were (5.72±0.31) % and (5.26±0.76) %, respectively. The IR of K562 and K562/ADR cells treated with 4μmol·L-1 VER were (4.10±0.45) % and (3.95±0.20) %, respectively. The IC50 of ADR combined with 4μmol·L-1 CH or 4μmol·L-1 VER in K562 cells were 0.44±0.02μmol·L-1 and 0.46±0.03μmol·L-1, respectively, without significantly differences from that of ADR alone at P>0.05. The IC50 of ADR combined with 4μmol·L-1 VER or 4μmol·L-1 CH in K562/ADR cells were 1.88±0.10μmol·L-1 and 6.27±0.17μmol·L-1, respectively, with significantly differences from that of ADR alone at P<0.05. The reversing fold was 2.23 and 7.43, respectively. The reversal effect of CH was higher than that of VER, and significantly different from them at P<0.05.3. Effect of CH on the accumulation of ADR in K562/ADR cellsThe K562/ADR cells were exposed to 5μg/ml ADR in the presence or absence of various concentrations of CH (0, 2, 4 and 8μmol·L-1) for 2 h, the intracellular ADR-associated MFI measured with flow cytometry were 3.1±0.3, 6.4±0.4,16.5±0.6 and 22.8±0.7, all groups were significantly different from each other at P<0.05.4. Effect of drugs on the function of P-glycoprotein in K562 and K562/ADR cellsThe uptake and efflux of rhodamine123 are used for the functional assay of P-gp in K562 and K562/ADR cells by measuring Rhol23-associated mean fluorescence intensity (MFI) using a flow cytometer. In P-gp accumulation analysis, the K562 or K562/ADR cells were exposed to 2μg/ml Rho123 in the presence or absence of various concentrations of CH (0, 2, 4 and 8μmol·L-1) or VER (4μmol·L-1 ) for 2 h, the Rhol23-associated MFI in K562 cells were81.5±0.4, 81.4±0.3, 81.7±0.3, 81.3±0.5 and 81.2±0.2, respectively; the MFI in K562/ADR cells were 16.9±0.6, 25.3±0.2, 44.7±0.3, 60.7±0.7 and 23.1±0.5, respectively, all K562/ADR groups were significantly different from each other at P<0.05, but all K562 groups were not significantly different from each other at P>0.05. In P-gp retention analysis, the K562 or K562/ADR cells were exposed to 2μg/ml Rhol23 for 2 h, then treated in the presence or absence of various concentrations of CH (0, 4 and 8μmol·L-1) or VER (4μmol·L-1) for 1 h, the MFI in K562 cells were 77.0±0.5, 77.2±0.1, 76.8±0.3 and 77.4±0.3, respectively; the MFI in K562/ADR cells were 4.0±0.1, 7.4±0.4, 13.7±0.2 and 4.9±0.1, respectively; All K562/ADR groups were significantly different from each other at P<0.05, but all K562 groups were not significantly different from each other at P>0.05.5. Effect of CH on the expression of P-gp in K562 and K562/ADR cellsThe expression of P-gp in K562 and K562/ADR cells was measured using a flow cytometer. The expression rate of P-glycoprotein in K562 cells was 0.3%, without significantly differences from K562 background cells (the positive rate was 0.3%) at P<0.05, suggesting there was no P-gp expression in K562 cells. The expression rate of P-glycoprotein in K562/ADR cells treated with various concentrations of CH (0, 2, 4 and 8μmol·L-1) or VER (4μmol·L-1) were (94.5±0.2)%, (94.6±0.1)%, (94.4±0.3)%, (94.5±0.1)% and (94.1±0.3)%, respectively, all groups were significantly different from each other at P<0.05, suggesting lower concentration of CH has no effect on the expression in K562/ADR cells.Conclusion1. CH partly reverses the drug resistance of K562/ADR cell line. The reversal effect of CH is stronger than that of VER.2. CH increases the accumulation of ADR in K562/ADR cells in a concentration-dependent manner.3. Rhol23 accumulation and retention presented a clear concentration relationship with the concentration of CH in K562/ADR cells, compared with the vehicle control, suggesting CH modulates intracellular Rho123 levels by inhibiting the function of P-gp. However, in K562 cells, there was no change in Rhol23 accumulation and retention in the presence of CH.4. There is overexpression of P-gp in K562/ADR cells, but no expression of P-gp in K562 cells. Lower concentration of CH has no effect on the expression of P-gp in K562/ADR cells.
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